Abstract The central enigma in tuberculosis treatment is the presence of persister cells?a drug tolerant phenotype characterized by low metabolic activity and very slow rate of growth. To significantly reduce the burden of tuberculosis, new therapeutic strategies that target persister cells are urgently required. This proposal suggests a new approach that targets the persistence phenotype by altering an environmental condition that favors Mtb survival. Specifically, we ask: is there a role for biofilm in persistence in tuberculosis, and if so, can we disrupt persistence by disrupting the biofilm? We propose to test the hypothesis that in a mouse model displaying human-like pathology, Mtb persister cells will be present in biofilm-like aggregates on DNA filaments in the acellular rim of necrotic granulomas, and that treatment of these infected mice with DNase will disrupt the aggregates and sensitize the Mtb cells to antimicrobial drugs, thereby shortening treatment time required for a durable cure. Our approach leverages historical strengths of the Trudeau Institute in mouse models of tuberculosis and advanced imaging technologies. We will utilize an innovative mouse model that displays human-like pathology to test the hypothesis that Mtb persister cells can be found in multicellular aggregates on extracellular DNA. We will employ fluorescent bacterial reporter strains that can be detected even when they lose acid-fast staining, as is typical of Mtb persister cells. In our first aim we setup and optimize the experimental system and test for the presence of Mtb persister cells associated with extracellular DNA. Then in aim 2 we will use the optimized system to address the fundamental goal of the proposal: test the efficacy of inhaled DNase as an adjunctive therapy for tuberculosis. We expect that DNase treatment will confer a significant benefit in more rapidly reducing bacterial burden than antitubercular drugs alone, providing justification for clinical testing of this novel adjunctive therapy.